Discovery of plant chemical defence mediated by a two-component system involving β-glucosidase in Panax species

Plants usually produce defence metabolites in non-active forms to minimize the risk of harm to themselves and spatiotemporally activate these defence metabolites upon pathogen attack. This so-called two-component system plays a decisive role in the chemical defence of various plants. Here, we discovered that Panax notoginseng, a valuable medicinal plant, has evolved a two-component chemical defence system composed of a chloroplast-localized β-glucosidase, denominated PnGH1, and its substrates 20(S)-protopanaxadiol ginsenosides. The β-glucosidase and its substrates are spatially separated in cells under physiological conditions, and ginsenoside hydrolysis is therefore activated only upon chloroplast disruption, which is caused by the induced exoenzymes of pathogenic fungi upon exposure to plant leaves. This activation of PnGH1-mediated hydrolysis results in the production of a series of less-polar ginsenosides by selective hydrolysis of an outer glucose at the C-3 site, with a broader spectrum and more potent antifungal activity in vitro and in vivo than the precursor molecules. Furthermore, such β-glucosidase-mediated hydrolysis upon fungal infection was also found in the congeneric species P. quinquefolium and P. ginseng. Our findings reveal a two-component chemical defence system in Panax species and offer insights for developing botanical pesticides for disease management in Panax species.

Generally, the data are described in a logic way and the manuscript is well written.However, to improve clarity of this submission, there are some points which need to take into consideration.
Regarding the specificity of PnGH1 1.Some fungi also have β-glucosidase, which has been proved to be able to catalyze the hydrolysis of ginsenosides.Have the authors identified whether the foliar pathogenic fungi used in this study contained β-glucosidases or not? 2. Three β-glucosidases, PnGH1, PnGH1-2 and PnGH-3, have been identified in P. notoginseng leaves.Have the authors confirmed the recombinant expression of PnGH1, PnGH1-2 and PnGH-3 by western blot?Besides, Supplementary Fig. 4 only showed the E. coli cells lysates before and after induction.The purification data of GH2 and 3 were missing.
3. In Fig. 3a and Fig. 3b, the SDS-PAGE showed two different molecular weight bands of PnGH1, please explain why.In addition, as PnGH1 is a 546-amino-acid protein, the SDS-PAGE results in both Fig. 3a and 3b were not consistent with its theoretical molecular weight.
4. The authors claimed that GH1 was a novel β-glucosidase with high enzyme stability, catalytic efficiency and hydrolytic regiospecificity.Have the authors compared GH1 with β-glucosidases in other species?
Regarding the antifungal activity of hydrolysis products 1.The efficacy control for hydrolytic products as well as carbendazim were compared at different dosage levels, which were 4 mg/mL (or 1mM) for hydrolytic products and 200 μM for carbendazim.As the dosage is very important for evaluating efficacy, the conclusion the authors made is not very reliable.
2. Is it possible that the hydrolysis products will also target beneficial fungi?Please provide experimental evidences.
Other minor comments 1.The "Abstract" does not briefly address the contents of this study.

Response Notes
Reviewer #1: Overall: Ma et al. discovered a β-glucosidase in Panax species that generates ginsenosides with improved antifungal activities.They first found that ginsenosides are hydrolyzed in the lesion area of P. notoginseng leaves with pathogenic fungal infection.The responsible β-glucosidase was identified by fractional purification and transcriptional analysis.The inhibitory activities of the hydrolyzed products were also analyzed.The manuscript is well-written and describes an important chemical defense system in Panax notoginseng, and I believe this work will be of highly interest for the community of plant natural product chemistry.Therefore, I recommend accepting this manuscript for publication in the journal.I have also listed minor questions for improvement.
− We sincerely appreciate the reviewer's positive and encouraging comments, which were of great help in revising the manuscript.

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We have addressed all the comments and concerns and revised the manuscript accordingly.

Is there any correlation between the expressions of the genes for the ginsenoside biosynthesis and β-glucosidase when infected by fungi?
− We sincerely appreciate the reviewer for the valuable and professional comments.
− To address this question, we collected the P. notoginseng leaves after being infected by the necrotrophic fungus M. acerina at different time points, including 0 h (control), 6 h, 12 h, 24 h, 48 h and 72 h.These samples were submitted for comparative transcriptome analysis.Key genes involved in the biosynthesis of ginsenosides in P. notoginseng (Fig. R1a) were searched through homology blast based on previously reported sequences (Luo et al., 2011;Niu et al., 2014;Xu et al., 2018;Jiang et al., 2022;Li et al., 2022) ).The expression profile of these genes and PnGH1 was carefully analyzed by constructing a z-Score heatmap (Fig. R1b) based on their FPKM values.As shown in Fig. R1b, key genes involved in ginsenoside biosynthesis were consistently upregulated after being infected by M. acerina, and most of them reached the highest expression level at 24 h.PnGH1 also exhibited an enhanced expression pattern in three samples (6 h-3, 12 h-3 and 24 h-2), with FPKM values almost six (6 h-3 and 12 h-3) and three times (24 h-2) higher compared to the control.These observations suggested that the biosynthesis of ginsenosides and PnGH1 gene were closely related to the chemical defence of P. notoginseng against M. acerina infection.These findings were added to the discussion section and Supplementary Fig. 22 in the revised manuscript.Besides, it was also noticed that after infection for 24 h, these genes were downregulated gradually to the normal level.The mechanism underlying this phenomenon is unknown.Supposedly, this may be physiologically required for specific homeostasis processes in plants.2. What is the estimated concentration of hydrolysis products in plants?Is it higher that the effective concentration determined by in vitro analysis?
− The levels of the hydrolysis products in decayed tissue gradually increased along with infection duration.According to the raw data of Fig. 2h and Fig. S2 − The total concentration of hydrolysis products at 5 DPI and 11 DPI are 48.36 ± 5.07 mg/g and 114.77 ± 12.06 mg/g of decayed leaves (wet weight), respectively.The hydrolysis products (TS-a) at 4 mg/mL exhibited obvious inhibitory effects against M. acerina growth in vitro and in vivo.Considering the density of the broth culture medium (0.61 g/mL), 4 mg/mL is equal to 6.56 mg/g of the medium.Thus, the concentration of hydrolysis products in decayed tissue is much higher than the effective concentration determined by in vitro test.

Can the authors exclude the possibility that metabolites of M. acerina activate the two-component system?
− We sincerely appreciate the reviewer for the careful review and valuable comments.
− In our study, the crude exoenzymes of M. acerina whose expression was induced by exposure to PNL tissue (both in vivo and in vitro) or pectin can activate the two-component system, rather than the broth culture of M. acerina.
− As shown in Figs.4a, 4c and 4f, M. acerina-inoculated medium without pectin or PNL tissue serves as in vitro control (Con_vitro), Con_vitro failed to produce any lesions on PNLs and trigger ginsenoside hydrolysis.Collectively, only induced exoenzymes isolated from M. acerina can activate the two-component system.

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We also exclude the possibility that M. acerina triggers ginsenoside hydrolysis.As shown in Fig. 2f, to determine whether a broth culture of a pathogenic fungus could cause the ginsenoside hydrolysis, Rb3 was spiked into an M. acerina culture, and the spiked culture was incubated for 11 days.The hydrolysis product Fd was not detected, and no significant difference in Rb3 content was observed at different intervals, indicating that enzymes secreted by M. acerina cannot trigger Rb3 hydrolysis.

Reviewer #2:
1.This is indeed an interesting study, but the novelty is restricted to the Panax system as a saponin two-component system was recently described by Lacchini et al, albeit with a nuclear located betaglucosidase.The major shortcoming of the paper is thus that it is focused entirely on the Panax system and fails to include state-of-the-art of other two-component systems regarding saponins nor to discuss the Panax system in regards to other beta-glucosidases in other two-component systems.
− We sincerely appreciate the reviewer for the careful review and valuable comments.
− Recently, Lacchini et al. reported the incredible and gorgeous work that Medicago truncatula, a model legume, has evolved a two-component system composed of a nucleolar-localized βglucosidase (G1) and triterpene saponins, acting as a saponin bomb.In this study, we first described a two-component chemical defence system involving chloroplast-localized βglucosidase and 20(S)-protopanaxadiol ginsenosides in Panax species.Lacchini's paper and our study adequately revealed that the saponin bomb model might be a widespread twocomponent system in many plant species.Additionally, Panax species encompasses several important ginseng species with high medicinal and economic values, including Panax notoginseng (Notoginseng), Panax ginseng (Asian Ginseng) and Panax quinquefolium (American Ginseng).Our findings are of great significance in revealing a chemical defence against fungal infection in Panax species and in developing botanical pesticides for disease management in Panax species, the highly valued medicinal plants.
− As suggested, the state-of-the-art of two-component systems involving triterpene saponins has been included in the revised manuscript.
2. Eg the take-home message of phylogenic analysis in supplementary figure 15 is not clear, nor how the tree was constructed.Perhaps focusing on a tree of plant beta-glucosidases and then include species names and substrate types would be more informative as a basis for an evolutionary perspective.I tried to locate the relevant beta-glucosidase from the Lacchini paper, but could not find it, as it requires that you know the accession member.
− We sincerely appreciate the reviewer for the careful review and constructive comments.
− As suggested, we have reconstructed the phylogenic tree as shown in Supplementary Fig. 15b.The take-home message of the phylogenic analysis was noted in the figure legends.The new tree was mainly focused on plant-originated β-glucosidases involved in plant defence.The listed sequences were selected from Lacchini's paper (Lacchini et al., 2023) and Morant's paper (Morant et al., 2008a).Species names and accession numbers of each sequence were provided in the legend.The substrate type of each sequence was displayed beside the enzyme on the right side, respectively.− We totally agree with the reviewer that chewing insects may destroy chloroplasts and activate the two-component system.We also agree to analyze regurgitate, rather than leaf tissue bitten by insects, to identify whether ginsenoside hydrolysis is associated with insect attack.
Unfortunately, in this study, the leaves bitten by herbivores were collected from open fields and further analyzed.We didn't further identify which type of insects are, chewing or sucking, due to the expertise limitation.Thus, the context regarding insect attacks has been removed from the revised manuscript to avoid confusing the readers.

Standard deviations for Km and Kcat should be rounded up/down.
− As suggested, we have made revisions accordingly in the main text and figures.
5. The authors discuss rates of turnover, but a rate would implies a time component, perhaps just calling it turnover in figure 1 would be more appropriate.
− We fully agree with the reviewer that "hydrolysis turnover" is more appropriate than "hydrolysis rate" in Figure 1.We have made revisions accordingly throughout the whole manuscript.
6. Some of the figures are very illustrative and helpful, but 3a is perhaps to generic.There are also numerous figures in the supplementary that are not referred to in the main text and that could me omitted.
spiked culture was incubated for 11 days.The hydrolysis product Fd was not detected, and no significant difference in Rb3 content was observed at different intervals, indicating that the broth culture of M. acerina cannot trigger the hydrolysis of Rb3.
2. Three β-glucosidases, PnGH1, PnGH1-2 and PnGH-3, have been identified in P. notoginseng leaves.Have the authors confirmed the recombinant expression of PnGH1, PnGH1-2 and PnGH-3 by western blot?Besides, Supplementary Fig. 4 only showed the E. coli cells lysates before and after induction.The purification data of GH2 and 3 were missing.
− We sincerely appreciate the reviewer for the careful review and valuable comments.

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The revised Supplementary Fig. 4 − Following your suggestion, the recombinant expression of PnGH1, PnGH2 and PnGH3 has been confirmed by western blot analysis as shown in the revised Supplementary Fig. 4c.The SDS-PAGE results of the recombinant expression of PnGH1, PnGH2 and PnGH3 in E. coli have also been updated in the revised Supplementary Fig. 4b showing cell lysates before and after induction, as well as the purified recombinant proteins.
3. In Fig. 3a and Fig. 3b, the SDS-PAGE showed two different molecular weight bands of PnGH1, please explain why.In addition, as PnGH1 is a 546-amino-acid protein, the SDS-PAGE results in both Fig. 3a and 3b were not consistent with its theoretical molecular weight.
− We sincerely appreciate the reviewer for the careful review and valuable comments.
− PnGH1 is a 546-amino-acid protein whose theoretical molecular weight is 61.85 kD.
− In Fig. 3b (Fig. 3a in the revised paper), the recombinant PnGH1 protein band was shown at a size larger than 70 kD.For heterologous expression, PnGH1 gene was constructed into pET-32a vector using EcoR I and Xho I as restriction sites.Based on the vector map, PnGH1 was expressed as a fusion protein consisting of 167 amino acids encoded from the vector at the Nterminal, whose theoretical molecular weight was approximately 17.9 kD.These amino acids include a Trx•tag, a His•tag and a S•tag sequences, which play crucial roles in facilitating the soluble expression of PnGH1 in E. coli.Thus, the fusion protein of PnGH1 was calculated to be around 79 kD, consistent with the observed band in Fig. 3b.We apologize for missing these details before, and the restriction sites have been noted in the experimental section in the revised version.
− Fig. 3a (Supplementary Fig. 3a in the revised paper) represents a protein band at the size between 40−50 kD, which is lower than the theoretical molecular weight of PnGH1.This protein was obtained through crude enzyme extraction followed by activity-guided purification procedures.A reasonable explanation was that some of the unstable segments in the protein were cleaved during the repeated purification processes or degraded by some chemicals or in vivo proteases after the cell was lysed.This speculation was made based on the following observation: (1) The three-dimensional structure of PnGH1 showed an obvious redundant region at the N-terminal 1-72 amino acids as shown in Fig. R2a.(2) Homology blast found that sequences in this region were not involved in the hydrolysis process.(3) A propeptide cleavage site was predicted between Lys72 and Arg73 with a score of 0.069 (Fig. R2b); (4) LC-MS proteomics analysis of the obtained protein combined with transcriptome sequence blast found PnGH1 showing the highest matching score which is far above other candidates (Supplementary Table 1), and their sequence alignment consistency begins at Arg73 of PnGH1 (labeled by a red triangle in Fig. R2c).To verify the speculation, we constructed the truncated PnGH1 (73-505 aa) and achieved its soluble expression in pET-32a vector.After removing the fusion tag through enterokinase cleavage, SDS-PAGE analysis displayed that the obtained protein represents a similar size, around ~45 kD (Fig. R2d and  R2e).Most importantly, this truncated protein also demonstrated robust hydrolysis activity towards Rb3 to generate Fd (Fig. R2f).These results strongly indicated that the protein obtained from activity-guided purification in Fig. 3a (Supplementary Fig. 3a in the revised paper) probably represents the core active unit of PnGH1 and thus exhibits a relatively smaller molecular weight in SDS-PAGE.4. The authors claimed that GH1 was a novel β-glucosidase with high enzyme stability, catalytic efficiency and hydrolytic regiospecificity.Have the authors compared GH1 with β-glucosidases in other species?
− We sincerely appreciate the reviewer for the professional suggestion.
− Regarding the stability of PnGH1, a typical (β/α)8 barrel fold, which contains eight parallel βstrands surrounded by eight helices, was observed in the three-dimensional structure of PnGH1.This structural type is a relatively stable structural fold (Czjzek et al., 2001).In addition, several salt bridges were observed in PnGH1 structure, which could contribute to the stability of PnGH1 (Sansenya et al., 2011).Furthermore, biochemical characterization has revealed that PnGH1 retains its activity across a wide pH range of 3.0 to 9.0 and a temperature range of 10°C to 80°C.Remarkably, more than 60% of its activity could be maintained under acidic conditions (pH value 4-5) or high temperatures (40-60 °C).Based on these observations, we described PnGH1 as a highly stable glucosidase.
− Regarding the catalytic efficiency of PnGH1 compared with other β-glucosidases, we have summarized the kinetic parameters of several reported β-glucosidases in Table R1, including β-glucosidases involved in plant defence (highlighted in blue), other plant-originated βglucosidases hydrolyzing natural glycosides (highlighted in yellow) and microbial βglucosidases hydrolyzing ginsenosides (highlighted in pink).From Table R1, it is easy to find that the KM values of most reported β-glucosidases are in the range of 1−5 mM.In contrast, the KM value of PnGH1 was determined to be 0.677 mM, indicating a relatively high substrate affinity towards ginsenoside Rb3.As for kcat and kcat/KM parameters, it appears that these parameters of currently reported glucosidases varied widely.The kcat/KM values of PnGH1 (5.8 s -1 •mM -1 ) showed no significant advantage compared with some other enzymes, such as GmICHG from Glycine max (3900 s -1 •mM -1 ), SG from Rauvolfia serpentine (106.4 s -1 •mM - 1 ), and BsAbfA from Bacilus subtilis (197.8 s -1 •mM -1 ).Accordingly, we consider PnGH1 as an efficient β-glucosidase rather than claiming it as "highly" efficient in the revised manuscript.
However, several points are worth further being noted here.Recently, Lacchini et al. (2023) reported a β-glucosidase MtG1 being involved in plant defence from Medicago truncatula, which also accepts triterpene saponins as the substrate.MtG1 was claimed as a highly efficient and fast β-glucosidase with a kcat/KM ratio of 26.299 s -1 •mM -1 .MeLinamarase (Keresztessy et al., 2001), a cyanogenic β-glucosidase involved in plant defense through cyanogenesis mechanism against small herbivores, exhibited a much lower kcat/KM value of 0.0246±0.0028s -1 •mM -1 toward its natural substrate linamarin.In our study, the kcat/KM value of PnGH1 (5.8 s -1 •mM -1 ) significantly surpasses that of MeLinamarase and is comparable to that of MtG1 in terms of magnitude, suggesting that the catalytic efficiency of PnGH1 is sufficient to play a crucial role in plant defence.− To summarize, the previous statement that "PnGH1 was a novel β-glucosidase with high enzyme stability, catalytic efficiency and hydrolytic regiospecificity" has been revised to "PnGH1 was a novel β-glucosidase with high enzyme stability and hydrolytic regiospecificity" in the discussion section.
5. The efficacy control for hydrolytic products as well as carbendazim were compared at different dosage levels, which were 4 mg/mL (or 1mM) for hydrolytic products and 200 μM for carbendazim.As the dosage is very important for evaluating efficacy, the conclusion the authors made is not very reliable.
− We sincerely appreciate the reviewer for the valuable and constructive comments.
− We agree with the review's opinion, and the relevant conclusion has been corrected accordingly."Given that hydrolysis products show the potent antifungal activity both in vitro and in vivo, our findings also provide new insights for developing green botanical pesticides for disease management in Panax species." 6.Is it possible that the hydrolysis products will also target beneficial fungi?Please provide experimental evidence.
− We sincerely appreciate the reviewer for the careful review and valuable comments.
− As suggested, the toxic effects of intact ginsenosides (TS-a) and their hydrolysis products (TS-b) against the growth of beneficial fungi were tested and compared in vitro.Two beneficial fungi, including Acremonium sp.D212, an endophytic fungus isolated from the buds of Panax notoginseng, and Saccharomyces cerevisiae (yeast strains WT HHY168), were used in this experiment.
− As shown in Fig. R1, TS-a exhibited significantly stronger inhibitory effects against the growth of both yeast and Acremonium sp.D212 at the applied concentrations than TS-b, indicating that refined extracts containing hydrolysis products are more toxic to a broad spectrum of fungi, including beneficial fungi, at the tested concentration ranges than intact ginsenosides.Furthermore, compared to M. acerina (Fig. 5 of the manuscript), hydrolysis products showed less inhibitory rate on Acremonium sp.D212 (25.2% vs 80.8% at the concentration of 4 mg/mL).Other minor comments 7. The "Abstract" does not briefly address the contents of this study.
− "Abstract" has been modified accordingly.Due to the 200-word limit, a general introduction and non-technical summary of the main results and their implications have been included.
− As suggested, the correction has been made accordingly."PnGH1" referred to genes were all in italics, while "PnGH1" represents proteins were all in normal representation.

Fig. R1 .
Fig. R1.(a) Biosynthesis of PPD-type ginsenosides in P. notoginseng (ginsenoside Rg3 as an example).Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; DMAPP, dimethylallyl diphosphate; IPP, isopentenyl diphosphate; FPP, farnesyl diphosphate.(b) Expression levels of PnGH1 and genes involved in ginsenosides biosynthesis after being infected by the necrotrophic fungus M. acerina at different time points.The expression values of each gene were transformed to z-scores based on the FPKM values and shown as a heatmap using Origin 2019 software.

Fig. R2 .
Fig. R2.(a) The three-dimensional structure of PnGH1 with 72 residues at N-terminal shown in blue.(b) Propeptide cleavage site prediction using DTU Health Tech's ProP tool.(c).Sequence alignment of PnGH1 and the tryptic peptide sequences obtained through LC-MS proteomics analysis.(d-e) SDS-PAGE analysis of active protein obtained from activityguided purification (d) and truncated PnGH1 (e).(f) HPLC analysis of the hydrolysis activity of PnGH1 and truncated PnGH1 towards Rb3.

Fig. R3
Fig. R3 In vitro antifungal activity of TS-a and TS-b against (a) yeast and (b-c) Acremonium sp.D212.b, Inhibitory effects on colony diameter at 13 DPI and (c) corresponding growth inhibition rate.

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As reported before, currently identified β-glucosidases involved in plant defence from monocots and dicots are separated, indicating an independent evolutionary development.Their substrate types mainly include cyanogenic glucosides, isoflavonoid glucosides, glucosinolates, etc.Only MtG1 was reported to hydrolyze triterpene saponins (3-Glc-28-Glcmedicagenic acid) in Medicago truncatula.However, it is phylogenetically located in the clade of isoflavone conjugate-hydrolyzing β-glucosidase and many cyanogenic glucoside glucosidases.Interestingly, β-glucosidases identified from Panax species in this paper, which also accepted triterpenoid saponins as substrate, were phylogenetically close to neither MtG1 nor other β-glucosidases.The four glucosidases from Panax species were clustered as a new subclade, indicating a distinct evolutionary branch.
3. The authors slow that tissue damage also cleavesthe saponins, but then they write that insects do not induce cleavage.It is not clear if the authors are discussing chewing or sucking insects nor how they did the experiments and what was actually analyzed.I would assume that chewing insects would destroy chloroplasts and release beta-glucosidases, at least there are examples of this in the literature.Normally the regurgitate is analyzed and not the leaf tissue that was taken a bite from.− Thank you very much for your professional and valuable comments that helped us improve this manuscript.